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Transcription initiation complex structures elucidate DNA opening

Nature volume 533, pages 353–358 (2016)Cite this article

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Abstract

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE ‘extended winged helix’ domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE ‘E-ribbon’ domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein–protein and protein–DNA contacts.

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Figure 1: Open complex structure at 3.6 Å resolution.
Figure 2: Basal factors position and retain DNA.
Figure 3: TFIIE architecture and interactions.
Figure 4: Closed complex cryo-EM structure.
Figure 5: Cleft clearance and DNA template loading.
Figure 6: Model for DNA opening during transcription initiation.

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Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Three-dimensional cryo-EM density maps of OC1, OC2, OC2-focused, OC3, OC3-focused, OC4, OC4-focused, OC5, and CC have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-3375, EMD-3376, EMD-3377, EMD-3378, EMD-3379, EMD-3380, EMD-3381, EMD-3382, and EMD-3383, respectively. Coordinate files of the OC and CC have been deposited in the Protein Data Bank under accession numbers 5FYW and 5FZ5. Coordinates and structure factors of the Pol II-Tfg1 peptide and the Pol II-TFIIF crystals have been deposited at the Protein Data Bank under the accession numbers 5IP7 and 5IP9.

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Acknowledgements

We thank C. Bernecky, W. Mühlbacher, S. Neyer, S. Sainsbury, and D. Tegunov for help and discussions; L. Larivière and L. Wenzeck for cloning and initial purification of TFIIE; W. Mühlbacher for initial cloning of TFIIA; S. Bilakovic for the modified pET-DUET-1 vector; J. Mahamid for help with data collection for the CC; K. Maier for help with yeast growth assays; M. Raabe and H. Urlaub for protein identification; K. Kinkelin for initial Pol II–TFIIF co-crystallization; and S. Hahn for providing the TFA1 yeast strain and the shuffle plasmid pSH810. C.P. (SFB860), M.H. (GRK1721), and P.C. were supported by the Deutsche Forschungsgemeinschaft, the Advanced Grant TRANSIT of the European Research Council, and the Volkswagen Foundation.

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Author notes

  1. C. Plaschka and M. Hantsche: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, 37077, Germany

    C. Plaschka, M. Hantsche, C. Dienemann, C. Burzinski & P. Cramer

  2. Max Planck Institute for Biochemistry, Am Klopferspitz 18, Martinsried, 82152, Germany

    J. Plitzko

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  1. C. Plaschka
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Contributions

C.P. designed and carried out high-resolution cryo-EM structure determinations of OC1–OC4. M.H. designed and carried out Pol II-TFIIF crystallographic analysis, and cryo-EM structure determinations of OC5 and CC. C.P. and M.H. designed and carried out functional assays. C.D. cloned and purified full-length TBP and TFIIA. C.D. and C.B. assisted with protein purification. J.P. supervised electron microscopy data collection. P.C. designed and supervised research. C.P., M.H., and P.C. prepared the manuscript.

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Correspondence to P. Cramer.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Modelling of open complex cryo-EM densities.

a, SDS–PAGE analysis of OC–cMed–Med1 complex after size-exclusion chromatography. Protein colours as in Fig. 1. Although core Mediator was required for stable association of TFIIE, it largely dissociated under cryo-EM conditions as observed previously19. Some remaining core Mediator was flexible and located as described previously19, but could not be included in further high-resolution analysis. b, Cryo-EM micrograph of the OC–cMed–Med1 complex. Scale bar, 50 nm. c, Ten representative reference-free 2D class averages of OC–cMed–Med1 reveal flexibility of the upstream DNA assembly including TFIIE (green arrow) and very weak density for core Mediator (orange arrow). Compare Extended Data Fig. 7a, c. d, Composite cryo-EM density of the OC shown in front and top views50. Colours indicate the cryo-EM densities used for modelling of the open complex (OC1, grey; OC2, green; OC2-focused, yellow; OC3, salmon; OC3-focused, blue; OC4 purple; OC4 round 2 class 2, light blue). Shown are the unsharpened cryo-EM densities. The percentage of particles from the full set of 257,259 that was used for the respective reconstruction is indicated. e, Composite cryo-EM density of the OC superimposed on a ribbon model of the OC, coloured as in Fig. 1. The composite cryo-EM density enabled modelling of the initiation factors and DNA. Our structure also enabled correction of the revised yeast initiation complex model obtained by Murakami et al. from cryo-EM at 6 Å resolution21, and we note the following differences between the structures, superimposed on Rpb1: (1) The TFIIF Tfg2 WH domain is rotated by ~180°, which is further inconsistent with nuclear magnetic resonance (NMR) data on the TFg2 WH–DNA interface68 and fits comparatively worse to protein–protein crosslinking data between the Tfg2 WH and Tfa2 WH1 (ref. 17). (2) Domains of TFIIE, except Tfa2 WH1, were placed incorrectly: Tfa1 eWH (rotation and translation into the E-linker density; 17 Å distance for helix α3 in our CC), Tfa1 E-ribbon (rotation and translation into E-linker density; 35 Å distance between the Zn atoms), and Tfa2 WH2 (~180° rotation). Further, the Tfa2 E-tether region was incorrectly assigned to density belonging to the Tfa1 eWH. The Tfa1 E-linker was not modelled. (3) The TFIIF Tfg1 arm was modelled into an empty space lacking density, and the Tfg1 helix α0 was absent. Our models of the TFIIF dimerization domain, Tfg2 linker, Tfg1 N terminus, and Tfg1 arm fit into densities from a recent study21, indicating the electron microscopic reconstruction is correct, but that the modelling was premature at the available resolution. f, Ribbon model of the OC coloured according to how different parts of the OC were modelled into the OC cryo-EM densities (see d). Regions with atomic (light blue) and backbone models (orange), and DNA (dark blue) are indicated. Views as in d. g, Representative regions of the sharpened cryo-EM densities OC1 (3.6 Å), OC2 (4.0 Å), and OC4 (3.9 Å) are shown with the underlying refined coordinate model. The OC1 density shows clear side-chain features for Rpb1 clamp helices α8 and α9 and Rpb2 β33, the OC4 density for Tfg1 β2 that is part of the dimerization domain, and the OC2 density for part of the Tfg2 linker. For OC nomenclature, see Extended Data Fig. 7. h, Fit of the TBP crystal structure (PDB: 1YTB)66 to the OC2 cryo-EM density, shown in a Pol II side view50. i, Fit of TFIIB N- and C-terminal cyclin domains, B-linker and B-reader, and B-ribbon elements to OC1 and OC2 cryo-EM densities. The B-linker element displays weak density, and the B-reader is not observed. j, Fit of the TFIIA crystal structure (PDB: 1YTF)23 to OC2-focused cryo-EM density in a Pol II top view50 (left). The four-helix bundle undergoes a minor rotation towards the β-barrel, and is apparently flexible (compare Extended Data Fig. 5e). Toa1 (middle) and Toa2 (right) subunit structures are shown. A large non-conserved insertion in Toa1 (Δ95–209), lacking in recombinant TFIIA (Methods), may affect the relative positioning of the four-helix bundle to the β-barrel. k, Fit of the TFIIF model to OC cryo-EM densities viewed from the top50. TFIIF dimerization domain and Tfg1 N-terminal region, arm, and charged helix elements are superimposed on the OC4 cryo-EM density. Tfg2 linker and WH domains are superimposed on OC2 and OC3-focused cryo-EM densities, respectively. Subunit architectures for Tfg1 (middle) and Tfg2 (right) subunits are shown, indicating disordered regions. Secondary structure elements were labelled according to the crystallographic model of the human RAP30–RAP74 heterodimer75. l, Fit of the TFIIE model to OC cryo-EM densities shown from the front50 (left). Models for Tfa1 eWH, E-linker and E-ribbon are superimposed onto OC1 and OC3 densities. Models for Tfa2 WH1 domain, Tfa2 WH2 and E-tether were fitted into OC3-focused and OC3 densities. Tfa1 (middle) and Tfa2 (right) subunits are shown, indicating disordered regions. The connectivity of the E-tether helices remains uncertain. m, Fit of promoter DNA to OC cryo-EM densities is shown in a side view50. A weak density for single-stranded template DNA contacts the Pol II fork loop 1, and is indicated by a blue arrow. Upstream and downstream DNA models are superimposed with OC3 and OC1 densities, respectively. The location of the Pol II active site magnesium ion is indicated.

Extended Data Figure 2 Details of TFIIF and the upstream DNA assembly.

a, View of the open complex from the side50. Pol II elements external 2 (dark green), lobe (yellow), protrusion (orange), Pol II subunit Rpb12 (dark blue) and basal factors TBP, TFIIB, and TFIIF are coloured as in Fig. 1. The remainder of the open complex is transparent. Green and purple boxes indicate the locations of TFIIB C-terminal cyclin and TFIIF dimerization domains, respectively. b, Interactions of TFIIB C-terminal cyclin domain with Pol II protrusion, Rpb12, Tfg2 linker and DNA. Colours as in a. c, Details of TFIIF dimerization domain interactions with Pol II external 2 and lobe50. d, Crystallographic analysis of the yeast-specific Tfg1 N-terminal region. Weak density for the Tfg1 N-terminal region was observed by cryo-EM (OC4 round 2 class 2) at low contour level (0.0155) close to Pol II elements external 1 and the hybrid binding region50 (left). X-ray analysis (right) of the corresponding peptide (Tfg1 F21–R35) enabled modelling and assignment of residue M27 (indicated with asterisk) owing to the anomalous signal. The Fo − Fc electron density map (grey, contour level 2.5σ), seleno-methionine anomalous difference Fourier (yellow, contour level 5σ), and final model in ribbon presentation (purple) are shown. The sequence of the synthetic peptide used for soaking into Pol II crystals is shown below. The modified methionine residue and predicted secondary structure are indicated. e, The Fo − Fc electron density maps obtained from soaking Pol II crystals with TFIIF (purple) and seleno-methionine labelled peptide (grey), respectively, show similar density in the same location on Pol II. f, The putative Tfg2 C terminus contacts TBP. Viewed from the side50. A tubular cryo-EM density from the OC3 map, low-pass filtered to 8 Å, emanates from the TFIIF Tfg2 WH–TFIIE Tfa2 WH1 density, and was tentatively assigned to the Tfg2 C-terminal region. The putative Tfg2 density reaches the TBP subunit, consistent with their suggested interaction29,76.

Extended Data Figure 3 Structure–function analysis of TFIIE and its interactions in the open complex.

a, The architectural model of TFIIE contains all regions required for viability in yeast16. A domain schematic (top) indicates the good overlap between modelled (dashed line) and functionally essential regions. Essential (grey), partially redundant Tfa2 WH1 and WH2 domains (blue), and non-essential elements (cyan) are indicated on the TFIIE model, shown in previously defined front and top views50 of Pol II. b, TFIIE sequence conservation. The sequence conservation among Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Gallus gallus, and Homo sapiens was mapped onto a ribbon representation of the TFIIE model. Highly, strongly, weakly and non-conserved residues are coloured in green, yellow, white, and grey, respectively. The location of a non-modelled helical density in the OC3 cryo-EM map, which may correspond to Tfa1 helix α7, is indicated. Views as in a. c, An additional density (green) in the OC3 cryo-EM map on top of the Tfa1 E-wing was tentatively assigned to Tfa1 helix α7 and this may stabilize the long β-hairpin. A front view is shown50. d, Tfa1–FeBABE cleavage sites in TFIIE16 are consistent with the TFIIE architecture. e, Tfa1– and Tfa2–FeBABE cleavage sites in the Pol II clamp16 and a protein–protein crosslink between Rpb1 K212 (Pol II clamp)–Tfa2 K277 (TFIIE E-tether)17 are consistent with the location of eWH and E-tether. f, Tfa2–Tfg2 protein–protein crosslinks17 are consistent with the Tfg2WH–Tfa2 WH1 architecture. g, The TFIIE mutations used for functional characterization were mapped onto a domain schematic and the model of TFIIE, shown in a front view50. h, Pulldown assays with recombinant TFIIE variants carrying mutations at the TFIIE–CC interface revealed that the E-ribbon is essential for TFIIE recruitment. For details of the TFIIE mutants, see g. Pulldowns were analysed by SDS–PAGE (Coomassie staining). To confirm the integrity of the purified TFIIE variants, 2 μg were analysed (left). Some minor contaminant and degradation bands of TFIIE are indicated by an asterisk. The bead elution from the pulldown assay is shown (middle), providing negative (no TFIIE) and positive (TFIIE) controls in the two leftmost lanes. The binding of all TFIIE variants to the CC was impaired compared to the wild-type protein, with the exception of the Tfa1(ΔE-wing) mutant, suggesting that all other interfaces contribute to TFIIE binding affinity. The most severe binding defect was observed upon mutation of three residues in the E-ribbon (Tfa1(L134/V137/L140)) to glutamate or alanine. This suggests that the E-ribbon is largely responsible for recruitment of TFIIE to the CC. The bead-only control (right) indicated that TFIIE and TFIIE variants did not show unspecific binding to the beads. i, Western blot analysis of the 3×Flag-tagged Tfa1 confirms specific immune-depletion of Tfa1 in the nuclear extract (NE), whereas levels of Pol II (Rpb3), TFIIB, and Histone H3 were unaffected. j, Yeast complementation assays were performed in triplicate experiments with wild-type TFA1, an empty vector, and TFA1 variants with mutations in the TFIIE eWH domain (N50E/K51E/T52E, N50A/K51A/T52A, and P56A/A59E/R62E in eWH helix α3, and ΔE-wing), or the E-ribbon(L134E/V137E/L140E) (see Methods). k, The long E-wing in the TFIIE subunit Tfa1 eWH is characteristic of WH domains involved in DNA strand separation77. The upstream edge of the transcription bubble and eWH domain are shown in a front view50 rotated by ~20° in the horizontal axis. Corresponding regions of human (Hs) Werner syndrome ATP-dependent helicase (WRN) WH (PDB: 2WWY) and RecQ1 WH (PDB: 3AAF) domains are shown.

Extended Data Figure 4 Closed complex and spontaneously formed open complex.

a, SDS–PAGE analysis of CC–cMed–Med1 complex after size-exclusion chromatography. Protein colours as in Fig. 1. b, Cryo-EM micrograph of the CC–cMed–Med1 complex. Scale bar, 50 nm. c, Ten representative reference-free 2D class averages of CC–cMed–Med1 reveal flexibility for the upstream complex. Core Mediator was not retained during cryo-EM analysis. d. Detailed view of the Pol II funnel helices in the CC (top) and OC5 (bottom) densities. e, Promoter sequences and differences in protein–DNA interactions are shown for the two distinct nucleic acid scaffolds used for preparation of closed and open complexes (compare Fig. 1d). Coloured bars indicate DNA–protein interaction. Solid, shaded, and empty circles respectively represent nucleotides included in the structure, excluded owing to weak cryo-EM density, or excluded owing to absence of cryo-EM density. Analogous yeast (black) and human (grey) numbering of promoter DNA is shown. The TATA-box sequence (red box) and HIS4-promoter sequence absent in the modified OC nucleic acid scaffold19 (grey box) are indicated. Protein–DNA interactions in the region covered by the light grey box are unchanged between CC and OC, and shown only for the OC for clarity. Unique and altered interactions are shown for each complex. DNA–TFIIEα photo-crosslinks, indicated by black asterisks, were observed in a closed but not open promoter state40 and are consistent with the CC model. f, Fit of TFIIE, Tfg2 WH and downstream DNA into CC density. Two rigid bodies were used for fitting: (i) Tfg2 WH and Tfa2 WH1 and (ii) Tfa2 WH2, eWH, E-linker and E-tether helices. Although the overall fit reflects density well, the eWH domain and its E-wing may be rotated further away from promoter DNA. g, Details on the location of downstream DNA (template, blue; non-template, light blue), Tfg2 WH, and Tfa2 WH1 and WH2 in the closed (dark colours) and open (light colours) complexes in the same view as in f. h, Cryo-EM density of OC5 and the OC ribbon model are shown in a front view50. The OC5 map shows weak density in regions of upstream assembly, TFIIE, and DNA that may be caused by increased flexibility owing to the heterogeneous population of spontaneously opened DNA. Colours as in Fig. 1. i, Fit of promoter DNA to cryo-EM densities of CC and OC5, shown in a side view50.

Extended Data Figure 5 Pol II cleft clearance, structural flexibility and rearrangements in the OC.

a, Pol II lid and fork loop 1 assume new conformations in the OC, clearing the Pol II cleft for loading of single-stranded template DNA. Arrows indicate the direction of movement of the two Pol II elements, and the template DNA loading path. The lid (dark red) in the open complex is moved in comparison to the lid of a Pol II–TFIIB ITC crystallographic study (PDB: 4BBS). Yellow and red boxes indicate zoomed-in regions of b and c, respectively. b, The movement in the Pol II lid leads to a steric clash with the TFIIB B-reader, observed in a Pol II–TFIIB ITC crystal (PDB: 4BBS), and facilitates its withdrawal in the open complex. In particular the lid residue F252 clashes with W63 and S67 of the B-reader. The OC1 cryo-EM density is shown for both lid and B-reader elements. c, The cryo-EM density of the OC1 reveals an ‘open’ Pol II fork loop 1 and a stably associated fragment of putative template DNA. The ‘open’ state of fork loop 1 provides additional space for loading of single-stranded template DNA past the Pol II rudder, towards the active site cleft. d, The position of the TFIIB N-terminal cyclin domain (light green) is altered in comparison to a Pol II–TFIIB ITC crystal structure15 (dark grey), but similar to its location in a cITC19 (light grey), probably owing to the presence of DNA. e, Flexibility of the upstream DNA assembly. The cryo-EM data of the OC was sorted on the basis of structural differences using an upstream assembly mask that included upstream DNA, TFIIA, TBP, and TFIIB cyclin domains (OC2 round 1, compare Extended Data Fig. 7c). Four of five resultant classes revealed different positions of the upstream complex, indicated here by fitted ribbon models of the OC. Previously defined front and side views50 are shown. Class 2 (middle) revealed the TFIIA four-helix bundle rotated by 85°, consistent with a high degree of flexibility. Class 4 represents the largest fraction of the data (31%), and gave a more defined density for the upstream complex, which was improved by further classification (Extended Data Fig. 7c). Class 5 presented with no density for the upstream complex or the Tfg2 linker, but did show density for the TFIIB B-ribbon and the TFIIF dimerization domain, suggesting that TFIIB and TFIIF remained bound to the complex. This is consistent with TFIIF-dependent association of the TFIIB-core domain with the Pol II wall27, and this apparently requires an ordered Tfg2 linker. f, The Rpb4–Rpb7 stalk adopts different positions in cITC, cITC-cMed, and OC. This suggests that Mediator and TFIIE may bind co-operatively. This is consistent with previous findings78 and with pulldowns (Extended Data Fig. 3h), which suggest that the TFIIE E-ribbon–stalk interface, which is important for TFIIE recruitment, is stabilized in the presence of Mediator.

Extended Data Figure 6 Pol II clamp positions and TFIIB B-reader mobility during DNA opening.

a, The yeast CC is shown from a side view50, indicating the path of DNA and location of TFIIE. The eye symbol (grey) indicates the point of view in b. b, The Pol II clamp may undergo transitions during DNA opening as indicated. The OC model of the Pol II clamp is shown superimposed on yeast CC (this study), and yeast OC (this study). The OC model Pol II clamp was rigid-body fitted to the human CC cryo-EM density20 (EMD-2306) and is superimposed. The view is from the front50. c, The TFIIB B-reader element shows strong density only in the ITC state, suggesting that its mobility in earlier states may be important for maintaining a cleared path for template DNA loading into the Pol II cleft. Ordering of the B-reader may further lead to stabilization of the upstream promoter assembly that is flexible in the OC (Extended Data Figs 5e, 7c). Cryo-EM densities for yeast CC (this work), OC5 (this work), OC (this work), and ITC (EMD-2785) complexes are superimposed on the TFIIB model (PDB: 4BBS for the B-linker and B-reader). As secondary structure elements could not be resolved in the human CC20, we excluded this cryo-EM density from comparison.

Extended Data Figure 7 Three-dimensional classification of cryo-EM data.

a, Three-dimensional image classification of the cryo-EM data set into eight classes using an initial OC reconstruction as the reference model, revealed heterogeneity. The percentage of single particles contributing to each class is provided. To help visualize structural differences, 3D reconstructions of the OC are coloured according to mobile regions: Pol II core, TFIIB B-ribbon (grey); upstream DNA, TFIIA, TBP, TFIIB cyclin domains, Tfg2 linker (green); TFIIF dimerization domain (purple); TFIIE except E-ribbon, Tfg2 WH (magenta); Pol II Rpb4–Rpb7 stalk and E-ribbon (blue); cMed–Med1 (yellow). b, Focused classification into five classes using a mask covering the Pol II stalk and E-ribbon. The resultant class 1 (OC1) was subsequently refined to 3.58 Å resolution (grey box) and revealed the location of the TFIIE E-ribbon. Colours as in a. c, Improvement of densities for Tfg2 linker, TFIIB, and TFIIE, through rounds of focused 3D classification using various masks. First, heterogeneity due to flexibility of upstream DNA and associated factors was overcome by applying a mask around this region (round 1). Focused refinement of the upstream DNA assembly of the resultant class 4 of round 1 (OC2-focused), improved the density quality for TFIIA (Extended Data Fig. 1j). Classification of the OC2-focused density revealed the upstream DNA complex (OC2) at 3.97 Å resolution (green box). Separate classification of class 4 of round 1 using OC, Pol II stalk and TFIIE E-ribbon, and TFIIE masks yielded class 1 of round 4 (OC3, magenta box) that contained a complete TFIIE density at a nominal resolution of 4.35 Å after 3D refinement (see Extended Data Fig. 8c). The small fraction of stably bound TFIIE is consistent with its reduced affinity to the pre-initiation complex79. Focused refinement of OC3 with a TFIIE–stalk mask (OC3-focused) improved density for Tfg2 WH and Tfa2 WH1 domains. Colours as in a. d, To improve the density of TFIIF dimerization domain and the Tfg1 arm, three rounds of classification using a TFIIF, TFIIF dimerization domain, and OC mask were employed. Class 2 of round 2 (cyan box) enabled fitting of the Tfg1 N-terminal peptide, which was resolved by X-ray analysis (Extended Data Fig. 2d, e). This class was further refined locally using a mask covering the TFIIF dimerization domain, and then classified with an OC mask, revealing class 6 of round 3 at 3.89 Å resolution after 3D refinement (purple box). Colours as in a. e, 3D classification of the CC cryo-EM data set into four classes, using an initial CC reconstruction as the reference model, revealed heterogeneity. Mobile regions in the reconstructions are highlighted: promoter DNA (blue), TFIIE (except E-ribbon), and Tfg2 WH (orange). Classifying the most populated classes from round 1 into three classes unexpectedly revealed open and closed promoter DNA states in the data set: CC (round 2, class 1) and OC5 (round 2, class 3). Class 3 of round 2 (OC5) was refined to 6.1 Å resolution (blue box). Class 1 from round 2 was further classified into three classes. The resultant class 3 of round 3 revealed density for closed downstream promoter DNA above the Pol II cleft, and TFIIE. The cryo-EM density for downstream DNA and TFIIE was improved by focused classification using two soft-edged masks. A mask covering the Pol II Rpb4–Rpb7 stalk yielded a class with better occupancy for the stalk (round 4, class 3), which was further sorted using a mask covering TFIIE and Tfg2 WH to improve their densities. Class 1 of round 5 was refined to 8.8 Å resolution (CC, orange box).

Extended Data Figure 8 Resolution of cryo-EM reconstructions.

a, Gold-standard FSC (left) of the OC1 cryo-EM single particle reconstruction (FSC = 0.143). Orientation distribution plot of all particles that contribute to the OC1 reconstruction (middle). The OC1 cryo-EM map is shown (right) from a front view50 and a central slice through the reconstruction, which are coloured by local resolution as described19. b, As in a but for the OC2 reconstruction. The gold-standard FSC for the density obtained from focused refinement (OC2-focused) with a soft mask around the upstream DNA assembly is indicated in grey (see Methods). The region masked for focused refinement is indicated with a grey outline on the cyro-EM map coloured by local resolution (right). c, As in b, but for the OC3 and OC3 focus-refined reconstructions. d, As in a, but for the OC4 and OC4 focus-refined reconstructions. e, As in a, but for the CC reconstruction. f, As in a, but for the OC5 reconstruction.

Extended Data Figure 9 Data collection, refinement statistics, and structure validation.

a, Cryo-EM data collection and refinement statistics of the OC structure. Different regions of the composite OC structure were refined into OC1, OC2, and OC4 maps as described (see Methods) to obtain an atomic model for 90% of the structure. b, Gold-standard FSC between the respective coordinate models and local regions of the OC1, OC2, and OC4 cryo-EM maps used for model refinement and between overall OC and CC models compared to OC3 (best TFIIE density) and CC cryo-EM maps. c, X-ray crystallographic data collection and refinement statistics.

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Plaschka, C., Hantsche, M., Dienemann, C. et al. Transcription initiation complex structures elucidate DNA opening. Nature 533, 353–358 (2016). https://doi.org/10.1038/nature17990

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